Oligonucleotides are synthesized routinely on solid supports using β-cyanoethyl phosphoramidite chemistry on a large scale for antisense therapeutics or on a small scale for diagnostic/molecular biology purposes. This chemistry was developed using silica based CPG beads as solid supports to facilitate deprotection and purification. Subsequently, this chemistry was adapted to synthesize oligonucleotides using rigid polystyrene based supports. In addition, large scale synthesis of oligonucleotides for use in antisense and related therapies has become increasingly important since FDA approval of a oligonucleotide analog for the treatment of CMV. Several other oligonucleotide analog are currently in clinical trials. Presently, kilogram quantities of oligonucleotides with either phosphate diester or phosphorothioated diester linkages are being synthesized using β-cyanoethyl phosphoramidite chemistry for clinical trials and other applications.
For pharmaceutical applications, the desired oligonucleotide must be separated from substantially all impurities generated during synthesis. Impurities are generally removed by reverse phase high pressure liquid chromatography (HPLC) and/or ion-exchange chromatography. However, those impurities which are structurally similar to the desired product are difficult to remove because their structural similarity to the desired product causes them to have a similar chromatographic mobility. Therefore, it is desirable to improve synthetic methodology so that impurities which have a similar structure to the desired product are not produced.